Biocatalystic synthesis of quinic acid and conversion to hydroquinone by recombinant microbes

ABSTRACT

A bioengineered synthesis scheme for the production of quinic acid from a carbon source is provided. Methods of producing quinic acid from a carbon source based on the synthesis scheme as well as conversion of quinic acid to hydroquinone are also provided.

RELATED APPLICATIONS

The present invention is a continuation of U.S. Ser. No. 11/184,707,filed Jul. 19, 2005, now issued as U.S. Pat. No. 7,642,083, which is acontinuation of U.S. Ser. No. 10/435,242, filed May 9, 2003, now issuedas U.S. Pat. No. 7,002,047, which is a continuation of U.S. Ser. No.09/427,394, filed Oct. 25, 1999, now issued as U.S. Pat. No. 6,600,077,which is a continuation-in-part of U.S. Ser. No. 09/240,441, filed Jan.29, 1999, now abandoned, all of which are hereby expressly incorporatedby reference in their entirety.

GOVERNMENT FUNDING

Work on this invention was sponsored in part by the United StatesDepartment of Agriculture Grant No. 95-37500-1930 and the NationalScience Foundation Grant No. CHE963368 amendment 002. The Government mayhave certain rights in the invention.

FIELD OF THE INVENTION

The present invention is related to the production of quinic acid andmore specifically, to methods of producing quinic acid and derivativesof quinic acid from the bioconversion of a carbon source.

BACKGROUND OF THE INVENTION

Quinic acid is an attractive chiral synthon with its highlyfunctionalized, six-membered carbocyclic ring and multiple asymmetriccenters. Both hydroquinone and benzoquinone, which are industriallyimportant organic compounds, can be derived by magnesium (IV) dioxideoxidation of quinic acid. Woskrensensky, A., Justus Liebigs Ann. Chem.27:257 (1838). Quinic acid is an important molecule utilized as anenantiomerically pure starting material for the synthesis of variousmolecules, many of which are biologically important. For example, quinicacid is a useful starting material for the synthesis of FK-506, animmune suppressive agent useful in preventing organ transplantrejection. Rao, A. V. R. et al., Tetrahedron Lett. 32:547 (1991).Additionally, quinic acid has been utilized in the synthesis of theneuraminidase inhibitor GS401 and GS4104, an important newpharmaceutical for the treatment of influenza. Barco, A. et al.,Tetrahedron Asymmetry 8:3515 (1997). It is also utilized as a convenientsource for the synthesis of many natural products that are otherwisedifficult to obtain (e.g., mycosporin andD-myo-inositol-1,4,5-triphosphate. White et al., J. Am. Chem. Soc.111(24):8970 (1989); Falck et al., J. Org. Chem. 54(25):5851 (1989),respectively. In addition, quinic acid is utilized as a food additive,resolving agent and is being used experimentally in optical materials.

Quinic acid has previously been isolated from natural sources (e.g.,cinchona bark, tobacco leaves, carrot leaves, etc.). However, the costof isolating quinic acid from such sources precludes its use as aneconomically viable starting material. Quinic acid has been synthesizedchemically, but such synthesis utilizes organic solvents, highlyreactive reagents and hazardous waste and as such is not environmentallydesirable. Therefore, there is a need for a cost effective,environmentally desirable method for the synthesis of quinic acid.

U.S. Pat. No. 5,798,236 describes a method for quinic acid productionthat uses a heterologous biocatalyst in which expression of quinatedehydrogenase from the Klebsiella pneumoniae qad gene in Escherichiacoli results in conversion of 3-dehydroquinic acid (DHQ) into quinicacid. Fermentation of this organism, E. coli AB2848aroD/pKD136/pTW8090A(ATCC 69086) produces a mixture of quinic acid, DHQ, and3-dehydroshikimic acid (DHS). While the relative molar ratio of thethree products varies with fermentation conditions, the molar ratio ofquinic acid to DHQ and to DHS fails to exceed 2:1:1. The appearance ofDHQ as a major byproduct is likely due to product inhibition of quinatedehydrogenase by quinic acid. Alternatively, the specific activity ofquinate dehydrogenase may be low due to poor expression of theKlebsiella gene in E. coli or due to instability of the plasmid carryingthe qad locus. The appearance of DHS may represent some instability inthe host organism itself.

Hydroquinone is a pseudocommodity chemical used in photographicdevelopers, polymerization inhibitors and antioxidants. Annualproduction of hydroquinones is in the 40,000-50,000 ton range.Krumenacher, L. et al. Hydroquinone is currently synthesized viahydroperoxidation of p-diisopropylbenzene as well as oxidation ofaniline or hydroxylation of phenol with hydrogen peroxide. U.S. Pat. No.5,798,236; Krumenacher, L. et al. Aniline, phenol, orp-diisopropylbenzene are produced from carcinogenic benzene startingmaterial, which is obtained from nonrenewable fossil fuel feedstocks.Methods have also been described for converting quinic acid tohydroquinone. U.S. Pat. No. 5,798,236. The quinic acid is over oxidizedto benzoquinone via hydroquinone, and the benzoquinone is then convertedback to hydroquinone.

It would thus be desirable to provide a method for the production ofquinic acid, which method utilizes a carbon source as a startingmaterial which can be derived from a renewable resource. It would alsobe desirable to provide a method for the production of quinic acid inwhich quinic acid is the major product at high concentrations comparedto by-products such as DHQ and DHS.

It would also be desirable to provide a method for the production ofhydroquinone from quinic acid. It would be further desirable for themethod to be inexpensive and utilize non-toxic and non-carcinogenicreactants. It would also be desirable for such a method to producehigh-purity hydroquinone in good yields without overoxidation of quinicacid to benzoquinone.

SUMMARY OF THE INVENTION

A bioengineered synthesis scheme for production of quinic acid from acarbon source is provided. In one embodiment, the bioconversion methodsof the present invention comprise the microbe-catalyzed conversion of acarbon source to quinic acid. As shown in the synthesis scheme of FIG.1, the microbe-catalyzed conversion step of the present inventionrequires three enzymes which are provided by a recombinant microbe. In apreferred embodiment, the recombinant microbe is Escherichia colidesigned to cause reduction of 3-dehydroquinate to quinic acid insteadof dehydration of 3-dehydroquinate to dehydroshikimate.

The biocatalytic synthesis of quinic acid provided herein isenvironmentally benign, economically attractive, and utilizes abundantrenewable sources as a starting material.

Also provided are methods for the conversion of quinic acid tohydroquinone. In one embodiment, quinic acid is initially oxidized to3,4,5-trihydroxycyclohexanone 1 (FIG. 4). In a preferred embodiment,quinic acid is oxidized by reaction with hyperchloric acid. Subsequentheating of 3,4,5-trihydroxycyclohexanone 1 yields hydroquinone.

Additional objects, advantages, and features of the present inventionwill become apparent from the following description and appended claims,taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The various advantages of the present invention will become apparent toone skilled in the art by reading the following specification andsubjoined claims and by referencing the following drawings in which:

FIG. 1 is a schematic illustrating the bioengineered synthesis scheme ofthe present invention for producing quinic acid;

FIG. 2 is a graph showing the production of quinic acid in comparison tocell dry weight;

FIG. 3 is a graph showing the equilibration of shikimic and quinic acidscatalyzed by SP1.1/pKD12.112;

FIG. 4 is a schematic illustrating the synthesis scheme of the presentinvention for converting quinic acid to hydroquinone;

FIG. 5 is a graph showing the production of hydroquinone over time withrelation to synthetic intermediates; and

FIG. 6 is a schematic illustrating the synthesis scheme for establishingthe synthetic route and relevant intermediates, wherein steps (a)through (e) are described as followed; (a) NaOCl, H₂SO₄, rt, 41%; (b)acetone, TsOH, 0° C., 59%; (c) Ac₂O, (i-Pr)₂NEt, DMAP, CH₂Cl₂, 0° C.,91%; (d) CF₃CO₂H/H₂O (2:1, v/v), 0° C., 43%; (e) (i) CH₃OH, Dowex® 50(H⁺), reflux; (ii) 2,3-butanedione, CH(OCH₃)₃, CH₃OH, CSA, reflux, 79%;(f) LiAlH₄, THF, 0° C., rt, 93%; (g) NaIO₄, phosphate buffer (pH 7), 0°C., rt, 72%; (h) Ac₂O, (i-Pr)₂NEt, DMAP, CH₂Cl₂, 0° C., 100%; (i)CF₃CO₂H/CH₂Cl₂/H₂O (9:1:1, v/v/v), 0° C., 75%.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A bioengineered synthesis scheme for the production of quinic acid froma carbon source is provided herein. Methods of producing quinic acidfrom a carbon source based on the synthesis scheme of FIG. 1 are alsoprovided. Methods for converting quinic acid to hydroquinone areprovided as well.

In one embodiment, a method is provided wherein the carbon source isconverted to quinic acid by a recombinant microbe. Manipulation of thecommon aromatic amino acid biosynthetic pathway of the microbe resultsin a significant production of quinic acid when the recombinant microbeis cultured in the presence of a carbon source. The carbon source isconverted to 3-deoxy-D-arabino heptulosonate-7-phosphate (DAHP) which issubsequently converted by 3-dehydroquinate synthase to 3-dehydroquinate(DHQ) which is then reduced to quinic acid by shikimate dehydrogenase(d, FIG. 1). The conversion of 3-dehydroquinate to quinic acid wasdiscovered when studying the production of shikimic acid in E. coliSP1.1/pKD12.112 from a carbon source as described in Specific Example 1.Quinic acid biosynthesis is surprising given the absence in E. coli ofquinate dehydrogenase which catalyzes the interconversion of3-dehydroquinate and quinic acid. This unprecedented activity ofshikimate dehydrogenase in the interconversion of 3-dehydroquinate andquinic acid has been confirmed by incubating 3-dehydroquinate withpurified shikimate dehydrogenase.

Quinic acid biosynthesis, while widespread in plants, has only beenobserved in a single microbe, E. coli AB2848aroD/pKD136/pTW8090A.Draths, K. M. et al., J. Am. Chem. Soc. 114:9725 (1992); and U.S. Pat.No. 5,798,236. This heterologous construct expresses quinatedehydrogenase encoded by the gad locus isolated from Klebsiellapneumoniae. Mitsuhashi, S. et al., Biochim. Biophys. Acta 15:268 (1954).Quinate dehydrogenase-catalyzed oxidation of quinic acid is driven bycatabolic consumption of the resulting 3-dehydroquinic acid via theβ-ketoadipate pathway in K. pneumoniae and other microbes. Reduction of3-dehydroquinic acid by quinate dehydrogenase dominates in E. coliAB2848aroD/pKD136/pTW8090A because of the absence of 3-dehydroquinicacid catabolism. Quinic acid synthesis in E. coli SP1.1/pKD12.112 thusimplicates the existence of an oxidoreductase which reduces3-dehydroquinic acid.

The bioconversion methods of the present invention are carried out underconditions of time, temperature, pH, nutrient type and concentration,aeration conditions, and controlled glucose concentrations, to providemaximal conversion of the carbon source to quinic acid. As described indetail in Specific Example 2, in a preferred embodiment, a fed-batchfermentor is used to convert the carbon source to quinic acid, followedby isolation of the quinic acid from the fermentation broth byion-exchange chromatography. The batch fermentor process andchromatography methods are also known to those skilled in the art.

As used herein, the phrase “carbon source” is meant to include biomassderived carbon sources including, but not limited to, xylose, arabinose,glycerol, glucose and the intermediates in the Krebs cycle (e.g.,dicarboxylic acids), either alone or in combination. In a preferredembodiment, the carbon source is glucose. The carbon source may bederived from renewable resources such as, without limitation, corn,sugar, beets and sugar cane.

In one embodiment, the recombinant microbe employed in the methods ofthe present invention is E. coli. In a preferred embodiment, the E. colicomprises a mutated aroD locus and an aroB cassette inserted into theserA locus. This recombinant E. coli, designated QP1.1, may furthercomprise a plasmid carrying aroF^(FBR), aroE and serA gene inserts. Theblocking or impeding of aroD-encoded 3-dehydroquinate dehydratase (c,FIG. 1) results in the accumulation of 3-dehydroquinate which issubsequently converted to quinic acid. It will be appreciated, however,that the aroD locus mutation is not essential and is employed to furtherprovide 3-dehydroquinate and to decrease the formation of3-dehydroshikimate acid. The aroB gene insert encodes 3-dehydroquinatesynthase (b, FIG. 1), increasing the production of 3-dehydroquinate. The3-dehydroquinate is converted into quinic acid by plasmid-localizedaroE-encoded shikimate dehydrogenase (d, FIG. 1).

In a preferred embodiment, the recombinant E. coli comprises plasmidpKD12.112 carrying aroF^(FBR), serA and aroE gene inserts. ThearoF^(FBR) gene insert encodes a mutant3-deoxy-D-arabino-heptulosonate-7-phosphate synthase isozyme (a, FIG. 1)insensitive to feedback inhibition by aromatic amino acids or otheraromatic molecules which increases carbon flow into the common aromaticamino acid biosynthetic pathway. Due to a mutation in the E. coligenomic serA locus required for L-serine biosynthesis, growth in minimalsalts medium and plasmid maintenance follows from expression ofplasmid-localized serA. The serA insert thus allows microbial growth inminimal salts medium, distinguishing the microbes containing the plasmidfrom non-plasmid containing microbes. The aroE gene insert encodes ashikimate dehydrogenase, increasing the production of quinic acid.Preferably, the aroE gene is from E. coli. More preferably, all theinserted genes are from E. coli, producing a homogenous recombinant E.coli.

In another embodiment, the aroF^(FBR), serA and/or aroE genes areinserted directly into the E. coli genome along with aroB. Such arecombinant E. coli would not require a plasmid to produce significantamounts of quinic acid.

The above-described preferred recombinant microbe of the presentinvention, E. coli QP1.1/pKD12.112, has been deposited with the AmericanType Culture Collection (ATCC), 10801 University Boulevard, Manassas,Va. 20110, under the terms of the Budapest Treaty, and has been accordedthe ATCC designation number 98904. The deposit will be maintained in theATCC depository, which is a public depository, for a period of 30 years,or 5 years after the most recent request, or for the effective life of apatent, whichever is longer, and will be replaced if the deposit becomesdepleted or nonviable during that period. Samples of the deposit willbecome available to the public and all restrictions imposed on access tothe deposit will be removed upon grant of a patent on this application.

The following table sets forth the three enzymes required for theconversion of glucose to quinic acid, the genes encoding same and theorigin of the genes in the exemplary recombinant microbes of the presentinvention.

TABLE 1 Enzyme^(†) Gene (origin) a) 3-deoxy-D-arabino-heptulosonicaroF^(FBR) (plasmid) acid 7-phosphate synthase b) 3-dehydroquinatesynthase aroB (additional copy inserted into genome) d) shikimatedehydrogenase aroE (plasmid) ^(†)Enzymes a), b) and d) correspond to a,b and d of FIG. 1.

Although E. coli is specifically described herein as the microbe forcarrying out the methods of the present invention, it will beappreciated that any microorganism such as the common types cited in theliterature and known to those skilled in the art, may be employed,provided the microorganism can be altered to effect the desiredconversion (e.g., carbon source to quinic acid, carbon source to3-dehydroquinate, 3-dehydroquinate to quinic acid, etc.) Thus, it isenvisaged that many types of fungi, bacteria and yeasts will work in themethods of the present invention. Such microorganisms may be developed,for example, through selection, mutation, and/or genetic transformationprocesses with the characteristic and necessary capability of convertingone constituent of the synthesis scheme of the present invention toanother. Methods for such development are well known to the skilledpractitioner.

In order to carry out the bioconversion methods of the presentinvention, a solution containing a carbon source is contacted with therecombinant microbe to form a bioconversion mixture which is maintainedunder appropriate conditions to promote the conversion of the carbonsource to the desired constituent, e.g., quinic acid. In a preferredembodiment, the bioconversion mixture is maintained at a temperature ofabout 30° C. to about 37° C. and a pH of about 6.5 to about 7.5. It ispreferred that the bioconversion mixture also contain other substancesnecessary to promote the viability of the recombinant microbes such asmineral salts, buffers, cofactors, nutrient substances and the like. Thebioconversion mixture is preferably maintained in a steady state underglucose limited conditions. In a preferred method the rate of glucoseaddition is determined by the level of dissolved oxygen concentration. Apreferred steady state over the course of fermentation is about 100 toabout 200 μmol glucose or a dissolved oxygen concentration of about 5%to about 35% air saturation. The more general requirements for themaintenance of viability of microorganisms are well known and specificrequirements for maintaining the viability of specific microorganismsare also well known as documented in the literature, or are otherwiseeasily determined by those skilled in the art. The quinic acid may thenbe recovered from the bioconversion mixture by methods known in the art(e.g., ion-exchange chromatography) and further purified byrecrystallization.

Novel methods for converting quinic acid to hydroquinone are alsoprovided. In one embodiment, a method is provided wherein oxidation ofquinic acid and heating of the oxidation product yields hydroquinone. Inone embodiment, quinic acid is oxidized to 3,4,5-trihydroxycyclohexanonewhich is then heated to produce hydroquinone (FIG. 4).

In one embodiment, the quinic acid is oxidized to3,4,5-trihydroxycyclohexanone wherein the oxidation is catalyzed by anoxidizing agent. In a preferred embodiment, the oxidizing agent oxidizesquinic acid to 3,4,5-trihydroxycyclohexanone but not to benzoquinone.Preferably, the oxidizing agent is chosen from the group comprisingHOCl, (H₂SO₄/NaOCl), AgNO₃, (NH₄)₂Ce(NO₃)₆, V₂O₅, NH₄VO₃ and2KHSO₅.KHSO₄ (oxone)/NaCl. More preferably, the oxidizing agent isselected from the group comprising HOCl or AgNO₃. Most preferably, theHOCl is formed in situ by acidifying commercial bleach (NaOCl) in theoxidation reaction mixture. The amount of oxidizing agent required ispreferably about 1.0 equivalents relative to quinic acid. Alternatively,the oxidizing agent is present in catalytic amounts of less than about1.0 equivalents along with at least 1.0 equivalents of a cooxidant. Forexample, 0.1 equivalents of AgNO₃ can effectively oxidize quinic acid inthe presence of 1.2 equivalents of K₂S₂O₈. While not wishing to be boundby theory, the cooxidant is present to regenerate the oxidizing agentsuch that only catalytic amounts of the oxidizing agent are required.

In another embodiment, the oxidation reaction is carried out in anaqueous quinic acid solution. In a preferred embodiment, the aqueousquinic acid solution is a purified quinic acid fermentation broth (seeSpecific Example 3). In an alternate embodiment, the aqueous quinic acidsolution is comprised of isolated quinic acid and water. The yield ofhydroquinone may be dependent on both the aqueous quinic acid solutionand the oxidizing agent. When the reaction was carried out in purifiedquinic acid fermentation broth, the yield of hydroquinone was 57%(mol/mol) with acidified commercial bleach, 55% (mol/mol) with(NH₄)₂Ce(NO₃)₆ and 64% (mol/mol) with V₂O₅ as the oxidants. However,when AgNO₃ was the oxidant, with K₂S₂O₈ as the cooxidant, the yield ofhydroquinone from purified quinic acid fermentation broth was only 35%(mol/mol) whereas the yield was increased to 80% (mol/mol) when theaqueous solution comprised purified quinic acid in water.

In a further embodiment, 3,4,5-trihydroxycyclohexanone is dehydrated toproduce hydroquinone. In a preferred embodiment, the3,4,5-trihydroxycyclohexanone is not isolated after the oxidationreaction. After oxidation of quinic acid to3,4,5-trihydroxycyclohexanone, the reaction mixture is then heated toproduce hydroquinone. When HOCl is the oxidizing agent, the oxidationreaction mixture is neutralized before heating. The hydroquinone canthen be purified from the reaction mixture by methods known in the art(e.g., extraction, flash chromatography).

In order to more fully demonstrate the advantages arising from thepresent invention, the following examples are set forth. It is to beunderstood that the following is by way of example only and is notintended as a limitation on the scope of the invention.

Specific Example 1 Synthesis of Quinic Acid by Shikimate Dehydrogenase

I. Results

Culturing SP1.1/pKD12.112 for 42 h with K_(c)=0.1 resulted in thesynthesis of 27.2 g/L of shikimic acid, 12.6 g/L of quinic acid, and 4.4g/L of 3-dehydroshikimic acid (DHS). DHS accumulation reflected theexpected feedback inhibition of shikimate dehydrogenase by shikimicacid. Draths, K. M. et al., J. Am. Chem. Soc. 114:9726 (1992); Dell, etal., J. Am. Chem. Soc. 115:11581 (1993). By contrast, quinic acidbiosynthesis was surprising given the absence in E. coli of quinic aciddehydrogenase which catalyzes 3-dehydroquinate and quinic acidinterconversion.

Quinic acid formation was further explored by collecting SP1.1/pKD12.112cells from the fermentor after 24 h. Washed cells were resuspended infresh fermentation medium containing shikimic acid and shaken. As shownin FIG. 3, formation of quinic acid (solid bars) and 3-dehydroshikimate(hatched bars) along with a corresponding decrease in shikimic acid(open bars) concentration indicated that SP1.1/pKD12.112 wastransporting shikimic acid into its cytosol. Brown, K. D. et al.,Biochim. Biophys. Acta 428:550 (1976). Since both shikimatedehydrogenase and 3-dehydroquinate dehydratase catalyze reversiblereactions, cytosolic shikimic acid could be converted back into3-dehydroquinate. Shikimate dehydrogenase might then play a dual role byalso catalyzing 3-dehydroquinate reduction.

Because of structural similarities between 3-dehydroshikimic and3-dehydroquinic acids (Scheme 1), purified E. coli shikimatedehydrogenase was incubated with 3-dehydroquinic acid. Quinic acidformation was observed. The Michaelis constant, K_(m)=1.2 mM, andmaximum velocity, V_(max)=0.096 mM-1min-1, for shikimatedehydrogenase-catalyzed reduction of 3-dehydroquinic acid to quinic acidcompares with K_(m)=0.11 mM and V_(max)=0.11 mM-1 min-1, for shikimatedehydrogenase-catalyzed reduction of 3-dehydroshikimic acid to shikimicacid.

II. Methods

General. For ¹H NMR quantitation of solute concentrations, solutionswere concentrated to dryness under reduced pressure, concentrated todryness one additional time from D₂O, and then redissolved in D₂Ocontaining a known concentration of the sodium salt of3-(trimethylsilyl)propionic-2,2,3,3-d₄ acid (TSP) purchased fromLancaster Synthesis Inc. Concentrations were determined by comparison ofintegrals corresponding to each compound with the integral correspondingto TSP (δ=0.00 ppm) in the ¹H NMR. All ¹H NMR spectra were recorded on aVarian VXR-300 FT-NMR Spectrometer (300 MHz).

Culture Medium. All medium was prepared in distilled, deionized water.M9 salts (1 L) contained Na₂HPO₄ (6 g), KH₂PO₄ (3 g), NaCl (0.5 g) andNH₄Cl (1 g). M9 minimal medium (1 L) consisted of 1 L of M9 saltscontaining D-glucose (10 g), MgSO₄ (0.12 g), thiamine hydrochloride(0.001 g), L-phenylalanine (0.040 g), L-tyrosine (0.040 g), L-tryptophan(0.040 g), p-hydroxybenzoic acid (0.010 g), potassium p-aminobenzoate(0.010 g), and 2,3-dihydroxybenzoic acid (0.010 g). Ampicillin was added(0.05 g/L) where indicated. Solutions of M9 salts, MgSO₄, and glucosewere autoclaved individually and then mixed. Aromatic amino acids,aromatic vitamins, and ampicillin were sterilized through 0.22-μmmembranes.

Fermentation medium (1 L) contained K₂HPO₄ (7.5 g), ammonium iron (III)citrate (0.3 g), citric acid monohydrate (2.1 g), L-phenylalanine (0.7g), L-tyrosine (0.7 g), L-tryptophan (0.35 g), and concentrated H₂SO₄(1.2 mL). Fermentation medium was adjusted to pH 7.0 by addition ofconcentrated NH4OH before autoclaving. The following supplements wereadded immediately prior to initiation of the fermentation: D-glucose (20or 23 g), MgSO₄ (0.24 g), p-hydroxybenzoic acid (0.010 g), potassiump-aminobenzoate (0.010 g), 2,3-dihydroxybenzoic acid (0.010 g), andtrace minerals including (NH₄)₆(Mo₇O₂₄)₅.4H₂O (0.0037 g), ZnSO₄.7H₂O(0.0029 g), H₃BO₃ (0.0247 g), CuSO₄.5H₂O (0.0025 g), and MnCl₂.4H₂O(0.0158 g). D-Glucose and MgSO₄ were autoclaved separately whilearomatic vitamins and trace minerals were sterilized through 0.22-μmmembranes.

Fermentations. Fermentations employed a 2.0 L working capacity B. BraunM2 culture vessel. Utilities were supplied by a B. Braun Biostat MD thatwas controlled by a DCU-1. Data acquisition utilized a Dell Optiplex Gs⁺5166M personal computer equipped with B. Braun MFCS/Win software.Temperature, pH, and glucose feeding were controlled with PID controlloops. Temperature was maintained at 33° C. pH was maintained at 7.0 byaddition of concentrated NH₄OH or 2 NH₂SO₄. Dissolved oxygen (D.O.) wasmeasured using a Mettler-Toledo 12 mm sterilizable O₂ sensor fitted withan Ingold A-type O₂ permeable membrane. D.O. was maintained at 10% airsaturation.

Inoculants were started by introduction of a single colony into 5 mL ofM9 medium containing ampicillin. The culture was grown at 37° C. withagitation at 250 rpm for 24 h and subsequently transferred to 100 mL ofM9 medium containing ampicillin. After growth at 37° C., 250 rpm for anadditional 12 h, the inoculant was ready for transfer into thefermentation vessel. The initial glucose concentration in thefermentation medium was 20 g/L for SP1.1/pKD12.112 runs and 23 g/L forQP1.1/pKD12.112 runs. Three staged methods were used to maintain D.O.levels at 10% air saturation during the course of run. With the airflowat an initial setting of 0.06 L/L/min, D.O. concentration was maintainedby increasing the impeller speed from its initial set point of 50 rpm toits preset maximum of 940 rpm. With the impeller constant at 940 rpm,the mass flow controller then maintained D.O. levels by increasing theairflow rate from 0.06 L/L/min to a preset maximum of 1.0 L/L/min. Atconstant impeller speed and constant airflow rate, D.O. levels werefinally maintained at 10% air saturation for the remainder of thefermentation by oxygen sensor-controlled glucose feeding. At thebeginning of this stage, D.O. levels fell below 10% air saturation dueto residual initial glucose in the medium. This lasted for approximately1 h before glucose (65% w/v) feeding started. The PID control parameterswere set to 0.0 (off) for the derivative control (T_(D)) and 999.9 s(minimum control action) for integral control (T₁). X_(p) was set to950% to achieve a K_(c) of 0.1.

Samples (10 mL) of fermentation broth were taken at 6 h intervals. Celldensities were determined by dilution of fermentation broth with water(1:100) followed by measurement of absorption at 600 nm (OD₆₀₀). Drycell weight (g/L) was obtained using a conversion coefficient of 0.43g/L/OD₆₀₀. The remaining fermentation broth was centrifuged for 4 minusing a Beckman microcentrifuge to obtain cell-free broth. Soluteconcentrations in the cell-free broth were determined by ¹H NMR.

Specific Example 2 Synthesis of Quinic Acid from Glucose

I. Results

Microbes were cultured under fed-batch fermentor conditions at 33° C.,pH 7.0, with dissolved oxygen maintained at a set point of 10% airsaturation. D-Glucose addition was controlled by dissolved O₂concentration and was a critical control parameter during syntheses ofshikimic and quinic acids. When dissolved oxygen levels exceeded the setpoint value indicating decreased microbial metabolism, the rate ofD-glucose addition was increased. When dissolved oxygen levels declinedbelow the set point value indicating increased microbial metabolism, therate of D-glucose addition was decreased.Proportional-integral-derivative control (PID) was used to control therate of D-glucose addition.

QP1.1/pKD12.112 was cultured for 60 h under fed-batch fermentorconditions at 37° C., pH 7.0, dissolved oxygen at 10% of saturation andan initial glucose concentration of 23 g/L. Extracellular accumulationof quinic acid began in early to mid log phase of microbial growth asrepresented by the open boxes in FIG. 2. The dry cell weight isrepresented by the solid line and closed circles. 3-Dehydroquinate wasefficiently converted to quinic acid as there was never a build-up ofthe intermediate (solid bars in FIG. 2). After 60 hours of cultivationwith K_(c)=0.1, E. coli QP1.1/pKD12.112 synthesized 60 g/L of quinicacid in 23% yield along with only 2.6 g of 3-dehydroquinate. Quinic acidwas isolated from the fermentation broth by ion-exchange chromatographyon AG1-x8 (acetate form) and Dowex 50 (H⁺ form) columns. Purified quinicacid was obtained by further recrystallization.

The yields of quinic acid obtained from culturing of the homologousconstruct QP1.1/pKD12.112 were significantly greater than those reportedfor the heterologous construct E. coli AB2848aroD/pKD136/pTW8090A.Cultures of QP1.1/pKD12.112 produced approximately 20 g/L of quinic acidafter 24 hours and 60 g/L after 60 hours (FIG. 2). In contrast, E. coliAB2848aroD/pKD136/pTW8090A only produced 10.7 g/L of quinic acid after24 hours. U.S. Pat. No. 5,798,236; Draths, K. M. et al., J. Am. Chem.Soc. 114:9725 (1992).

Microbial synthesis of quinic acid may supplant isolation of thishydroaromatic from plant sources which has limited its syntheticutility. At the same time, increased availability of quinic acid mayportend wider utilization of this hydroaromatic. The theoretical maximumyield of microbial synthesis of quinic acid is 43% from D-glucose.Draths, K. M. et al., J. Am. Chem. Soc. 117:2395 (1995). Comparison withthe yields achieved thus far for microbial synthesis of quinic acid(23%) along with the apparent lack of toxicity of this hydroaromatictowards the microbial biocatalyst, suggest that sizable increases inyields and titers are possible.

II. Methods

General. For ¹H NMR quantitation of solute concentrations, solutionswere concentrated to dryness under reduced pressure, concentrated todryness one additional time from D₂O, and then redissolved in D₂Ocontaining a known concentration of the sodium salt of3-(trimethylsilyl)propionic-2,2,3,3-d₄ acid (TSP) purchased fromLancaster Synthesis Inc. Concentrations were determined by comparison ofintegrals corresponding to each compound with the integral correspondingto TSP (6=0.00 ppm) in the ¹H NMR. All ¹H NMR spectra were recorded on aVarian VXR-300 FT-NMR Spectrometer (300 MHz).

Culture Medium. All medium was prepared in distilled, deionized water.M9 salts (1 L) contained Na₂HPO₄ (6 g), KH₂PO₄ (3 g), NaCl (0.5 g) andNH₄Cl (1 g). M9 minimal medium (1 L) consisted of 1 L of M9 saltscontaining D-glucose (10 g), MgSO₄ (0.12 g), thiamine hydrochloride(0.001 g), L-phenylalanine (0.040 g), L-tyrosine (0.040 g), L-tryptophan(0.040 g), p-hydroxybenzoic acid (0.010 g), potassium p-aminobenzoate(0.010 g), and 2,3-dihydroxybenzoic acid (0.010 g). Ampicillin was added(0.05 g/L) where indicated. Solutions of M9 salts, MgSO₄, and glucosewere autoclaved individually and then mixed. Aromatic amino acids,aromatic vitamins, and ampicillin were sterilized through 0.22-μmmembranes.

Fermentation medium (1 L) contained K₂HPO₄ (7.5 g), ammonium iron (III)citrate (0.3 g), citric acid monohydrate (2.1 g), L-phenylalanine (0.7g), L-tyrosine (0.7 g), L-tryptophan (0.35 g), and concentrated H₂SO₄(1.2 mL). Fermentation medium was adjusted to pH 7.0 by addition ofconcentrated NH₄OH before autoclaving. The following supplements wereadded immediately prior to initiation of the fermentation: D-glucose (23g), MgSO₄ (0.24 g), p-hydroxybenzoic acid (0.010 g), potassiump-aminobenzoate (0.010 g), 2,3-dihydroxybenzoic acid (0.010 g), andtrace minerals including (NH₄)₆(Mo₇O₂₄).4H₂O (0.0037 g), ZnSO₄.7H₂O(0.0029 g), H₃BO₃ (0.0247 g), CuSO₄.5H₂O (0.0025 g), and MnCl₂.4H₂O(0.0158 g). D-Glucose and MgSO₄ were autoclaved separately whilearomatic vitamins and trace minerals were sterilized through 0.22-μmmembranes.

Fermentation. Fermentation employed a 2.0 L working capacity B. Braun M2culture vessel. Utilities were supplied by a B. Braun Biostat MD thatwas controlled by a DCU-1. Data acquisition utilized a Dell Optiplex Gs⁺5166M personal computer equipped with B. Braun MFCS/Win software.Temperature, pH, and glucose feeding were controlled with PID controlloops. Temperature was maintained at 33° C. pH was maintained at 7.0 byaddition of concentrated NH₄OH or 2 NH₂SO₄. Dissolved oxygen (D.O.) wasmeasured using a Mettler-Toledo 12 mm sterilizable O₂ sensor fitted withan Ingold A-type O₂ permeable membrane. D.O. was maintained at 10% airsaturation.

An inoculant was started by introduction of a single colony into 5 mL ofM9 medium containing ampicillin. The culture was grown at 37° C. withagitation at 250 rpm for 24 h and subsequently transferred to 100 mL ofM9 medium containing ampicillin. After growth at 37° C., 250 rpm for anadditional 12 h, the inoculant was ready for transfer into thefermentation vessel. The initial glucose concentration in thefermentation medium was 23 g/L. Three staged methods were used tomaintain D.O. levels at 10% air saturation during the course of run.With the airflow at an initial setting of 0.06 L/L/min, D.O.concentration was maintained by increasing the impeller speed from itsinitial set point of 50 rpm to its preset maximum of 940 rpm. With theimpeller constant at 940 rpm, the mass flow controller then maintainedD.O. levels by increasing the airflow rate from 0.06 L/L/min to a presetmaximum of 1.0 L/L/min. At constant impeller speed and constant airflowrate, D.O. levels were finally maintained at 10% air saturation for theremainder of the fermentation by oxygen sensor-controlled glucosefeeding. At the beginning of this stage, D.O. levels fell below 10% airsaturation due to residual initial glucose in the medium. This lastedfor approximately 1 h before glucose (65% w/v) feeding started. The PIDcontrol parameters were set to 0.0 (off) for the derivative control(T_(D)) and 999.9 s (minimum control action) for integral control (T₁).X_(p) was set to 950% to achieve a K_(c) of 0.1.

Samples (10 mL) of fermentation broth were taken at 6 h intervals. Celldensities were determined by dilution of fermentation broth with water(1:100) followed by measurement of absorption at 600 nm (OD₆₀₀). Drycell weight (g/L) was obtained using a conversion coefficient of 0.43g/L/OD₆₀₀. The remaining fermentation broth was centrifuged for 4 minusing a Beckman microcentrifuge to obtain cell-free broth. Soluteconcentrations in the cell-free broth were determined by ¹H NMR.

Purification of Quinic Acid from Fermentation Broth. The fermentationbroth (1100-1200 mL) was centrifuged at 14000 g for 20 min and the cellswere discarded. The resulting supernatant was refluxed for 1 h, cooledto room temperature, and the pH adjusted to 2.5 by addition ofconcentrated H₂SO₄. After centrifugation at 14000 g for 20 min, a clearyellow solution was poured away from the cellular debris and adjusted topH 6.9 by addition of concentrated NH₄OH. The solution was combined with10 g of Darco KB-B activated carbon, swirled at 50 rpm for 1-2 h, andthen filtered through Whatman 5 filter paper. Filtered material waswashed with an additional 300 mL of water.

Following treatment with activated carbon, the solution was slightlygray in color. Addition of glacial acetic acid to a final concentrationof 25% afforded a clear, yellow solution which was then eluted through acolumn of AG1-x8 (acetate form, 5 cm×20 cm) at 4° C. Following elutionof the column with an additional 400 mL of 25% aqueous acetic acid, thecombined eluents were passed through a column of Dowex 50 (H⁺ form, 5cm×20 cm) at 4° C. which was then washed with 400 mL of 25% aqueousacetic acid. The eluents off the cation exchange column were combinedand were concentrated to dryness by rotary evaporation, leaving a hardwhite solid (80% recovery through this step). Recrystallization fromethanol afforded a fine, white powder (52% recovery based on quinic acidquantified in crude fermentation broth).

Specific Example 3 Synthesis of Hydroquinone from Quinic Acid withAcidified NaOCl

I. Results

Hydroquinone was synthesized from quinic acid as shown in the syntheticscheme of FIG. 4. Quinic acid was oxidized to3(R),4(S),5(R)-trihydroxycyclohexanone 1 upon reaction with acidified(H₂SO₄) commercial bleach (NaOCl). After quenching excess oxidant withisopropanol E, heating 1 afforded hydroquinone via intermediacy ofcyclohexanones 2 and 3 (FIG. 4). The progress of the reaction uponheating with respect to time is shown in FIG. 5. Astrihydroxycyclohexanone 1 (solid diamonds in FIG. 5) decreases, initialincreases are observed for cyclohexanones 2 and 3 (open squares andshaded triangles, respectively, in FIG. 5). As the reaction progresses,the intermediates decrease and hydroquinone is formed (-x- in FIG. 5).

II. Methods

General Chemistry. ¹H NMR spectra were recorded at 300 MHz on a VarianGemini-300 spectrometer. Chemical shifts for ¹H NMR spectra are reported(in parts per million) relative to internal tetramethylsilane (Me₄Si,δ=0.0 ppm) with CDCl₃ as a solvent, and to sodium3-(trimethylsilyl)propionate-2,2,3,3-d₄ (TSP, δ=0.0 ppm) when D₂O wasthe solvent. ¹³C NMR spectra were recorded at 75 MHz on a Gemini-300spectrometer. Chemical shifts for ¹³C NMR spectra are reported (in partsper million) relative to CDCl₃ (δ=77.0 ppm) or internal acetonitrile(CH₃CN, δ=3.69 ppm) in D₂O.

Purification of quinic acid from fermentation broth. Quinic acid wassynthesized by the methods of Specific Example 2. Following removal ofcells, 1 L of crude culture supernatant containing quinic acid (82.9 g,0.431 mol, 1000 mL) was refluxed for 1 h. After the solution cooled toroom temperature, concentrated sulfuric acid was added to a final pH of2.5. The resulting solution was then centrifuged and filtered, thesupernatant was decolorized by stirring with charcoal (20 g) at roomtemperature for 2 hours. Finally, the clear solution was passed throughDowex 50 (H⁺) column at 4° C. to afford an aqueous quinic acid solution.(81.5 g, 0.424 mol, 98%)

Synthesis of hydroquinone. To a stirred solution of the purified quinicacid (81.5 g, 0.424 mol) in 1120 mL broth at room temperature was addedcommercial bleach (1800 mL) and H₂SO₄ (178 mL, 2 M final concentration)dropwise over a 1 h period. The mixture was stirred for an additional 2hours. Isopropanol (130 mL) was added to quench unreacted HOCl. Theresulting solution which contained3(R),4(S),5(R)-trihydroxycyclohexanone 1 (50.1 g, 0.343 mol, 81%) washeated to reflux under an argon atmosphere for 10 h. After cooling toroom temperature, hydroquinone was extracted into t-butyl methyl ether(4×500 mL) and the combined organic layers were dried over MgSO₄.Charcoal (20 g) was added to the solution which was then stirred for 10minutes and subsequently filtered through Celite. The filtered residuewas washed with t-butyl methyl ether (200 mL). The filtrates wereconcentrated in vacuo to obtain hydroquinone as a brown solid in 77%yield. Sublimation of the isolated material yielded hydroquinone aswhite solid in 93% yield. The overall yield from quinic acid is 57%.

Synthesis of 3(R),4(S),5(R)-trihydroxycyclohexanone (1). To a stirredsolution of quinic acid (20 g, 0.104 mol) in 120 mL water at roomtemperature was added commercial bleach (NaOCl, 442 g) and H₂SO₄ (41.6mL, 2 M final concentration). After the mixture was stirred for 3 hours,isopropanol (15.9 mL) was added and the solution was stirred for anadditional 30 minutes. The resulting solution was neutralized andconcentrated, and EtOH/EtOAc (300 mL, 1:1, v/v) was added to theresulting slurry and the mixture was stirred for 30 minutes. Resultinginorganic salts were removed by filtration and the resulting solutionwas concentrated to dryness and compound 1 was obtained as a white solidafter being purified by flash chromatography (6.15 g, 41%). ¹H NMR (D₂O)δ 4.27-4.31 (m, 1H), 4.15 (ddd, J=8.2, 8.2, 5.1 Hz, 1H), 3.97 (dd,J=7.5, 2.7 Hz, 1H), 2.75-2.83 (m, 2H), 2.50-2.65 (m, 2H); ¹³C NMR (D₂O)δ 215.5, 75.7, 71.4, 48.4, 48.3.

Synthesis of 3,4-O-isopropylidene-3(R),4(S),5(R)-trihydroxycyclohexanone(4). Trihydroxycyclohexanone 1 (3.0 g, 20.5 mmol) was dissolved in 60 mLacetone, and TsOH (30 mg, 0.158 mmol) was added. The solution wasstirred vigorously under an argon atmosphere at room temperature for 9hours. Removal of the solvent afforded a yellow oil which was purifiedby flash chromatography. Compound 4 (FIG. 6) was obtained as whitecrystals (2.24 g, 59%). ¹H NMR (CDCl₃) δ 4.70-4.74 (m, 1H), 4.32 (ddd,J=7.2, 2.1, 2.1 Hz, 1H), 4.24 (dd, J=6.3, 2.7 Hz, 1H), 2.82 (dd, J=17.8,3.9 Hz, 1H), 2.70-2.72 (m, 1H), 2.65-2.66 (m, 1H), 2.46 (dm, 1H, J=17.8Hz), 2.24 (b, 1H), 1.45 (s, 3H), 1.37 (s, 3H); ¹³C NMR (CDCl₃) δ 208.0,108.8, 74.9, 72.2, 68.2, 41.6, 40.1, 26.4, 23.8.

Synthesis of 3,4-O-Isopropylidene-4(S),5(R)-dihydroxy-2-cyclohexen-1-one(5). To a solution of 4 (2.0 g, 10.8 mmol) in CH₂Cl₂ (8 ml) at 0° C. wasadded 4-(dimethylamino)pyridine (20 mg), diisopropylethylamine (3.75 mL,21.5 mmol) and acetic anhydride (1.22 mL, 12.9 mmol). After stirring for3 hours at 0° C., the reaction mixture was washed with saturated aqueousNaHCO₃ (2.5 20 ml), and the NaHCO₃ solution was extracted with CH₂Cl₂(4×40 mL). The organic layer was dried over MgSO₄ and concentrated todryness to afford a pale yellow solid. Kugelrohr distillation gavecompound 5 (FIG. 6) (1.65 g, 91%) as a colorless oil which crystallizedas white solid. ¹H NMR (CDCl₃) δ 6.65 (dd, J=10.2, 3.6 Hz, 1H), 6.04 (d,J=10.2 Hz, 1H), 4.71 (m, 2H), 2.93 (dd, J=17.7, 2.7 Hz, 1H), 2.70 (dd,J=17.7, 3.9 Hz, 1H), 1.39 (s, 3H), 1.38 (s, 3H); ¹³C NMR (CDCl₃) δ208.4; 108.7; 74.8; 72.2; 68.0; 41.5; 40.1; 26.3; 23.8.

Synthesis of 4(S),5(R)-dihydroxy-2-cyclohexen-1-one (3). Compound 5 (0.5g, 2.98 mmol) was dissolved in CF₃CO₂H/H₂O (2:1, v/v, 25 mL) and themixture was stirred at 0° C. for 20 minutes. The solvent was removed invacuo, and the compound was purified by flash chromatograph to givecompound 3 (FIG. 6) (0.16 g, 43%) as colorless oil, which crystallizedinto white solid. ¹H NMR (D₂O) δ 6.97 (dm, J=10.2 Hz, 1H), 6.11 (dm,J=10.2 Hz, 1H), 4.69-4.72 (m, 1H), 4.39-4.41 (m, 1H), 2.82 (ddd, J=17.0,3.3, 0.8 Hz, 1H), 2.72 (dd, J=17.0, 5.0 Hz, 1H); ¹³C NMR (D₂O) δ 204.4;154.2; 151.5; 72.4; 70.3; 45.6.

Synthesis of compound 7. BBA-protected methyl quinate 6 (FIG. 6) (4.0 g,12.5 mmol) was dissolved in 50 mL dry THF at 0° C. and LiAlH₄ (1.42 g,37.5 mmol) was added portionwise. The mixture was stirred for 1 hour at0° C. then warmed to room temperature. After 10 hours, all the startingmaterial has been consumed. The resulting mixture was cooled to 0° C.and excess hydride was quenched by careful, successive addition of water(1.4 mL), 15% aqueous NaOH (1.4 mL) and water (4.2 mL). Celite (8.0 g)was added and the slurry was stirred for 2 hours. The alumina salt wasseparated by vacuum filtering through a pad of Celite, and washed withhot ethyl acetate (100 mL). The filtrate was then concentrated todryness and purified by flash chromatography to afford the desiredproduct 7 (FIG. 6) as a white foam (3.4 g, 93%). ¹H NMR (CDCl₃) δ4.26-4.35 (m, 1H), 4.21-4.22 (m, 1H), 3.55 (dd, J=10.2, 2.7 Hz, 1H),3.40 (dd, J=33.2, 11.0 Hz, 1H), 3.27 (s, 3H), 3.25 (s, 3H), 3.13 (b,1H), 2.44 (b, 1H), 2.24 (dm, J=14.7 Hz, 1H), 1.93-1.98 (m, 1H),1.38-1.52 (m, 2H), 1.33 (s, 3H), 1.30 (s, 3H); ¹³C NMR (CDCl₃) δ 100.3,99.7, 74.1, 73.4, 70.4, 69.5, 62.9, 48.0, 47.9, 37.7, 36.0, 17.9, 17.6.

Synthesis of BBA-protected 3,4,5-trihydroxycyclohexanone (8). Triol 7(FIG. 6) (2.5 g, 8.56 mmol) was dissolved in 50 mL phosphate buffer (pH7) and the solution cooled to 0° C. Sodium periodate (2.74 g, 12.8 mmol)was added in portions. After the addition, the ice-bath was removed andthe mixture was stirred at room temperature for 1 hour. The aqueousmixture was then extracted with ethyl acetate (4×50 mL), and thecombined organic phase was dried with MgSO₄ and filtered through Celite.The solvent was removed in vacuo and compound 8 (FIG. 6) was obtained asa white powder. (1.61 g, 72%). ¹H NMR (CDCl₃) δ 4.23-4.33 (m, 2H), 3.89(dd, J=10.2, 2.4 Hz, 1H), 3.31 (s, 3H), 3.24 (s, 3H), 2.61-2.69 (m, 2H),2.45-2.54 (m, 2H), 1.82 (b, 1H), 1.35 (s, 3H), 1.31 (s, 3H); ¹³C NMR(CDCl₃) δ 205.5, 100.1; 99.2; 72.2; 67.6; 63.2; 48.1; 47.9; 46.2; 44.7;17.7; 17.5.

Synthesis of BBA-protected dihydroxycyclohexenone (9). To a solution ofthe β-hydroxy ketone 8 (FIG. 6) (1.01 g, 3.85 mmol) in CH₂Cl₂ (10 mL) at0° C. was added 4-(dimethylamino)pyridine (9.4 mg, 0.077 mmol),diisopropylethylamine (1.34 mL, 7.7 mmol) and acetic anhydride (0.44 mL,4.6 mmol). After stirring at 0° C. for 6 hours, the solution was washedwith saturated aqueous NaHCO₃ and the NaHCO₃ wash solution was extractedwith CH₂Cl₂ (4×40 ml). The organic layer was dried with MgSO₄ andconcentrated to dryness. Purification by flash chromatography affordedcompound 9 (FIG. 6) as a white solid (0.94 g, 100%). ¹H NMR (CDCl₃) δ6.87 (dd, J=10.2, 1.8 Hz, 1H), 6.01 (dd, J=10.2, 2.4 Hz, 1H), 4.51 (dt,J=9.0, 2.1 Hz, 1H), 4.05 (m, 1H), 3.33 (s, 3H), 3.27 (s, 3H), 2.74 (dd,J=16.5, 13.2 Hz, 1H), 1.37 (s, 3H), 1.34 (s, 3H); ¹³C NMR (CDCl₃) δ196.8; 148.5; 130.1; 100.8; 99.7; 69.2; 8.0; 48.2; 48.1; 42.0; 17.7;17.6.

Synthesis of 4(5), 5(S)-dihydroxy-2-cyclohexen-1-one (2). Theα,β-unsaturated ketone 9 (FIG. 6) (0.344 g, 1.42 mmol) was stirred in amixture of trifluoroacetic acid (18 mL), CH₂Cl₂ (2 mL) and water (2 mL)for 30 minutes at 0° C. Solvents were removed in vacuo. The residue wasazeotroped with toluene (20 mL) and purified by flash chromatography togive compound 2 (FIG. 4) as a white solid (0.137 g, 75%). ¹H NMR (D₂O) δ7.04 (dd, J=10.2, 2.4 Hz, 1H), 6.08 (dm, J=10.2 Hz, 1H), 4.45 (dm, J=8.4Hz, 1H), 3.99-4.07 (m, 1H), 2.82 (ddd, J=16.4, 4.8, 1.2 Hz, 1H), 2.58(dd, J=16.4, 11.9 Hz, 1H); ¹³C NMR (D₂O) δ 204.8, 156.1, 131.4, 74.4,74.3, 46.5.

Specific Example 4 Synthesis of Hydroquinone from Quinic Acid with AgNO₃

To quinic acid broth (106 mL containing 1.84 g, 9.6 mmol quinic acid),K₂S₂O₈ (3.11 g, 11.5 mmol) and AgNO₃ (0.163 g, 0.96 mmol) was added. Thesolution was stirred vigorously at 50° C. under an argon atmosphere for4 h and then heated to reflux for an additional 9 h. Extraction withethyl acetate (4×50 mL) followed by drying over MgSO₄ and concentrationafforded a brown solid. Purification by flash chromatography yieldedhydroquinone (0.37 g, 35%) as a white solid.

The foregoing discussion discloses and describes merely exemplaryembodiments of the present invention. One skilled in the art willreadily recognize from such discussion, and from the accompanyingdrawings and claims, that various changes, modifications and variationscan be made therein without departing from the spirit and scope of theinvention as defined in the following claims.

All references cited herein are incorporated by reference as if fullyset forth. In addition, U.S. Ser. No. 09/240,440, entitled “BiocatalyticSynthesis Of Shikimic Acid,” filed Jan. 29, 1999, is also expresslyincorporated by reference.

1. A method for production of quinic acid comprising: (A) providing oneor more enzymes selected from: (i) a 3-deoxy-D-arabino-heptulosonicacid-7-phosphate (DAHP) synthase enzyme that is insensitive to feedbackinhibition by aromatic amino acids, (ii) a 3-dehydroquinate (DHQ)synthase enzyme, and (iii) a shikimate dehydrogenase enzyme; wherein atleast one of said enzymes is a recombinant enzyme, wherein the DAHPsynthase, DHQ synthase, and shikimate dehydrogenase are encoded by E.coli aroF^(FBR) , E. coli aroB, and E. coli aroE genes, respectively;and (B) allowing the one or more enzymes to catalyze one or morereactions to synthesize quinic acid from erythrose-4-phosphate (E4P) andphosphoenolpyruvate (PEP); thereby producing quinic acid.
 2. The methodaccording to claim 1, wherein the quinic acid is produced by arecombinant microbe, wherein (a) the one or more enzymes are produced bythe recombinant microbe, (b) at least one of the enzymes are encoded byrecombinant DNA, (c) the recombinant microbe comprises an inactivatingmutation in an endogenous DNA encoding a dehydroquinate dehydratase, (d)the recombinant microbe is capable of producing quinic acid from acarbon source by using all three enzymes, and (e) the E4P and PEP areproduced by the recombinant microbe, wherein the recombinant microbe iscultured to allow the one or more enzymes to catalyze the one or morereactions to synthesize quinic acid from the E4P and PEP.
 3. A methodfor production of hydroquinone from quinic acid, comprising: (A)providing quinic acid produced by the method according to claim 1, andan oxidizing agent; (B) contacting the quinic acid with the oxidizingagent to produce 3,4,5-trihydroxycyclohexanone; and (C) heating the3,4,5-trihydroxycyclohexanone to produce hydroquinone.
 4. The methodaccording to claim 3, wherein the oxidizing agent is selected from thegroup consisting of HOCl, AgNO₃, (NH₄)₂Ce(NO₃)₆, V₂O₅, NH₄VO₃, and2KHSO₅.KHSO₄.K₂SO₄/NaCl.
 5. The method according to claim 3, wherein thequinic acid is produced by a recombinant microbe comprising: (A) theDAHP synthase enzyme that is insensitive to feedback inhibition byaromatic amino acids, (B) the DHQ synthase enzyme, and (C) the shikimatedehydrogenase enzyme, wherein the DAHP synthase, DHQ synthase andshikimate dehydrogenase are encoded by the E. coli aroF^(FBR) , E. coliaroB, and E. coli aroE genes, respectively; wherein (a) at least one ofsaid enzymes are encoded by recombinant DNA, (b) the recombinant microbecomprises an inactivating mutation in an endogenous DNA encoding adehydroquinate dehydratase, and (c) the recombinant microbe is capableof producing quinic acid from a carbon source by using all threeenzymes, wherein the recombinant microbe is cultured to allow all threeenzymes to catalyze the reactions to synthesize quinic acid.
 6. Themethod according to claim 2, wherein the recombinant microbe is arecombinant E. coli.
 7. The method according to claim 1, wherein the E.coli aroE gene is the same aroE gene found in plasmid pKD12.112.
 8. Themethod according to claim 2, wherein the inactivating mutation is in anE. coli aroD gene.
 9. The method according to claim 8, wherein therecombinant microbe is E. coli QP1.1.